Exoenzyme S (ExoS) is secreted by Pseudomonas aeruginosa and like many bacterial toxins has an ADP-ribosyltransferase activity. Although animal model studies suggest that ExoS contributes to P. aeruginosa virulence, it is not classified as a toxin since its targets and mode of action in the mammalian host remain unknown. Difficulties in understanding the function of ExoS in vivo relate directly to an inability to develop a system that allows the cellular effects of ExoS to be detected and analyzed. These difficulties are believed to reflect the inability of the soluble form of ExoS to bind and enter eukaryotic cells in a manner similar to other bacterial toxins. Homology recognized between ExoS and Yersina Yops virulence determinants supports that ExoS is secreted via a type Ill secretory process which requires direct contact between the bacterium and eukaryotic cell for the efficient translocation of the protein. Based on this premise, we have developed a bacterial/eukaryotic co-culture system that allows ExoS producing bacteria to contact target cells and their effect on cellular function assessed. Using this system we have been able to detect and quantify an inhibitory effect of ExoS on cell proliferation and viability. Studies described in this proposal will use this system to determine: 1) the cell-type and/or cell condition specificity of ExoS effects on cellular function, 2) the in vivo substrate specificity of the ExoS ADP-ribosyltransferase activity, 3) the contribution of the ExoS domains to its function, and 4) the functional inter-relatedness of 49 kDa ExoS and its close relative 53 kDa ExoT. Proteins secreted via the type lll secretory process appear to be involved in an intimate sensing process between the bacterium and target cell which results in the direct biochemical exchange of signals. It is anticipated that ExoS, and its close relative ExoT, will play a similar sensing role in P. aeruginosa. P. aeruginosa functions in environmental recycling and as an opportunistic pathogen, in both instances sensing compromised or decaying cell matter. The hypothesis of this proposal is that ExoS plays a central role in the opportunistic sensing process of P. aeruginosa, and that an understanding of the effects of ExoS and ExoT on cellular function is critical to understanding mechanisms of P. aeruginosa pathogenesis and adaptability.